Anthocyanins abrogate glutamate-induced AMPK activation, oxidative stress, neuroinflammation, and neurodegeneration in postnatal rat brain.

BACKGROUND: Glutamate-induced excitotoxicity, oxidative damage, and neuroinflammation are believed to play an important role in the development of a number of CNS disorders. We recently reported that a high dose of glutamate could induce AMPK-mediated neurodegeneration in the postnatal day 7 (PND7) rat brain. Yet, the mechanism of glutamate-induced oxidative stress and neuroinflammation in the postnatal brain is not well understood. Here, we report for the first time the mechanism of glutamate-induced oxidative damage, neuroinflammation, and neuroprotection by polyphenolic anthocyanins in PND7. METHODS: PND7 rat brains, SH-SY5Y, and BV2 cells treated either alone with glutamate or in combination with anthocyanins and compound C were examined with Western blot and immunofluorescence techniques. Additionally, reactive oxygen species (ROS) assay and other ELISA kit assays were employed to know the therapeutic efficacy of anthocyanins against glutamate. RESULTS: A single injection of glutamate to developing rats significantly increased brain glutamate levels, activated and phosphorylated AMPK induction, and inhibited nuclear factor-E2-related factor 2 (Nrf2) after 2, 3, and 4 h in a time-dependent manner. In contrast, anthocyanin co-treatment significantly reduced glutamate-induced AMPK induction, ROS production, neuroinflammation, and neurodegeneration in the developing rat brain. Most importantly, anthocyanins increased glutathione (GSH and GSSG) levels and stimulated the endogenous antioxidant system, including Nrf2 and heme oxygenase-1 (HO-1), against glutamate-induced oxidative stress. Interestingly, blocking AMPK with compound C in young rats abolished glutamate-induced neurotoxicity. Similarly, all these experiments were replicated in SH-SY5Y cells by silencing AMPK with siRNA, which suggests that AMPK is the key mediator in glutamate-induced neurotoxicity. CONCLUSIONS: Here, we report for the first time that anthocyanins can potentially decrease glutamate-induced neurotoxicity in young rats. Our work demonstrates that glutamate is toxic to the developing rat brain and that anthocyanins can minimize the severity of glutamate-induced neurotoxicity in an AMPK-dependent manner.

Neuroprotective effect of N-acyl 5-hydroxytryptamines on glutamate-induced cytotoxicity in HT-22 cells.

Some endocannabinoids have been known to express anti-inflammatory and antioxidant actions independent of cannabinoid receptors. In this respect, we investigated whether N-acyl 5-hydroxytryptamines (5-HTs) might prevent against glutamate-induced oxidative cytotoxicity in HT-22 cells, and attempted to elucidate the mechanism for their cytoprotective action. N-acyl 5-HTs with palmitoyl, stearoyl, arachidonoyl or docosahexaenoyl chain expressed a remarkable protective effect on glutamate-induced cytotoxicity. Additionally, glutamate-induced oxidative stress, represented by the increase of reactive oxygen species level and the reduction of glutathione level, was prevented markedly by N-acyl 5-HTs at submicromolar levels. Further, N-palmitoyl 5-HT, the most cytoprotective, enhanced antioxidant defense by up-regulating the expression of antioxidant enzymes such as heme oxygenase-1, glutamate-cysteine ligase catalytic subunit or NAD(P)H quinine oxidoreductase-1 in the presence or absence of glutamate. Consistent with this, N-palmitoyl 5-HT stimulated nuclear translocation of Nrf2 in early phase (2 h), and this effect was remarkably suppressed by inhibitors of PI3K, PDK-1, Akt or p38 MAPK. Additionally, N-palmitoyl 5-HT suppressed glutamate-induced activation of ERK in late phase (12 h), but not in early phase (2 h), presumably supporting the implication of MEK/ERK pathway in glutamate-induced cytotoxicity. Collectively, it is suggested that N-acyl 5-HTs may attenuate glutamate-induced cytotoxicity via the activation of PI3K/PDK-1/Akt- and p38 MAPK-dependent Nrf2 signaling in early phase as well as the suppression of MEK/ERK pathway in late phase.

Seventy day safety assessment of an orally ingested, l-glutamine-containing oat and yeast supplement for horses.

We describe a safety assessment of an oral supplement designed to nutritionally support the gastrointestinal system of horses. The supplement comprised a mixture of essential (l-threonine) and conditionally essential (l-glutamine) amino acids, polar lipids, oat bran rich in beta glucans and yeast extract. Young (1-2years) horses of both sexes were allocated to control (n=7) and treatment groups (n=7) and studied for 9weeks. Horses in the treatment group received the supplement daily for 8weeks. After 8weeks of supplementation, horses were studied for one additional week. Outcome measures included body mass, weight gain, results of clinical examination, hematology and plasma chemistry. There were no adverse events associated with supplementation and horses in both groups showed normal weight gain, clinical signs, hematology and chemistry. l-Glutamine, which is not yet listed as GRAS, was considered with respect to its potential for nutritional support and safety when ingested orally. It is concluded that this oral supplement, when ingested by horses at twice the recommended daily level, was safe and does not pose a health risk when used in accordance with good feeding practice.

The anti-leprosy drug clofazimine reduces polyQ toxicity through activation of PPARγ.

BACKGROUND: PolyQ diseases are autosomal dominant neurodegenerative disorders caused by the expansion of CAG repeats. While of slow progression, these diseases are ultimately fatal and lack effective therapies. METHODS: A high-throughput chemical screen was conducted to identify drugs that lower the toxicity of a protein containing the first exon of Huntington's disease (HD) protein huntingtin (HTT) harbouring 94 glutamines (Htt-Q94). Candidate drugs were tested in a wide range of in vitro and in vivo models of polyQ toxicity. FINDINGS: The chemical screen identified the anti-leprosy drug clofazimine as a hit, which was subsequently validated in several in vitro models. Computational analyses of transcriptional signatures revealed that the effect of clofazimine was due to the stimulation of mitochondrial biogenesis by peroxisome proliferator-activated receptor gamma (PPARγ). In agreement with this, clofazimine rescued mitochondrial dysfunction triggered by Htt-Q94 expression. Importantly, clofazimine also limited polyQ toxicity in developing zebrafish and neuron-specific worm models of polyQ disease. INTERPRETATION: Our results support the potential of repurposing the antimicrobial drug clofazimine for the treatment of polyQ diseases. FUNDING: A full list of funding sources can be found in the acknowledgments section.

Effects of glutamine supplementation on kidney of diabetic rat.

Glutamine is the most important donor of NH(3) in kidney playing an important role in acid-base buffering system. Besides this effect, glutamine presents many other relevant functions in the whole body, such as a precursor of arginine in adult and neonates. In addition to these effects, some studies have shown that glutamine can potentiate renal disease. In the present study, the effect of short-term treatment (15 days) with glutamine on control and diabetic rats was investigated. Using biochemical, histological and molecular biology analysis from control and diabetic rats we verified that glutamine supplementation increase in pro-inflammatory interleukins (IL)-1beta and IL-6 content in renal cortex and induce alteration in glomerular characteristics. This study showed that short-term treatment with glutamine in association with increased glucose levels could cause important alterations in glomerular morphology that may result in fast progression of kidney failure.

L-Glutamine-induced apoptosis in microglia is mediated by mitochondrial dysfunction.

Under physiological conditions, astrocytes take up L-glutamate from the synaptic gap, metabolize it to L-glutamine and return it to neurons, where L-glutamine is metabolized to L-glutamate and stored in neurotransmitter vesicles. However, under pathological conditions, such as hepatic failure, L-glutamine and ammonium are elevated globally in the brain. The Trojan horse hypothesis of L-glutamine toxicity assumes that intramitochondrial hydrolysis of L-glutamine enhances ammonium locally and leads to mitochondrial dysfunction. In the present study, we show that exposure of murine primary microglia as well as of the microglial cell-line BV-2 to L-glutamine promotes chromatin condensation and formation of crescent-like structures in the nucleus. Furthermore, L-glutamine induced an increase in annexin-V labelling, cell shrinkage (apoptotic volume decrease), cell fragmentation and formation of apoptotic bodies. Inhibition of the phosphate-activated glutaminase with 6-diazo-5-oxo-L-norleucine suppressed chromatin condensation and annexin-V labelling in L-glutamine-exposed cells. In addition, inhibition of the glutamine synthetase with L-methionine sulfoximine suppressed chromatin condensation and annexin-V labelling in ammonium-exposed cells. L-glutamine and ammonium enhanced production of reactive oxygen species, as detected with CM-H(2)DCFDA. Apoptosis, induced by L-glutamine, was inhibited either by the radical scavenger alpha-tocopherol or by the nitric oxide synthase blocker N (G)-methyl-L-arginine. Cyclosporin A, a ligand of the permeability transition pore complex component cyclophilin D, prevented L-glutamine-triggered apoptosis. Furthermore, blockade of caspase-9 activity with Z-LEHD-FMK prevented L-glutamine-triggered apoptosis. Taken together, our results indicate that hydrolysis of l-glutamine and, accordingly, accumulation of ammonium in mitochondria induce the intrinsic pathway of apoptosis, characterized by mitochondrial dysfunction and activation of caspase-9, which activates caspase-3.

γ-Glutamyltransferase enzyme activity of cancer cells modulates L-γ-glutamyl-p-nitroanilide (GPNA) cytotoxicity.

L-γ-Glutamyl-p-nitroanilide (GPNA) is widely used to inhibit the glutamine (Gln) transporter ASCT2, but recent studies have demonstrated that it is also able to inhibit other sodium-dependent and independent amino acid transporters. Moreover, GPNA is a well known substrate of the enzyme γ-glutamyltransferase (GGT). Our aim was to evaluate the effect of GGT-mediated GPNA catabolism on cell viability and Gln transport. The GGT-catalyzed hydrolysis of GPNA produced cytotoxic effects in lung cancer A549 cells, resulting from the release of metabolite p-nitroaniline (PNA) rather than from the inhibition of Gln uptake. Interestingly, compounds like valproic acid, verapamil and reversan were able to increase the cytotoxicity of GPNA and PNA, suggesting a key role of intracellular detoxification mechanisms. Our data indicate that the mechanism of action of GPNA is more complex than believed, and further confirm the poor specificity of GPNA as an inhibitor of Gln transport. Different factors may modulate the final effects of GPNA, ranging from GGT and ASCT2 expression to intracellular defenses against xenobiotics. Thus, other strategies - such as a genetic suppression of ASCT2 or the identification of new specific inhibitors - should be preferred when inhibition of ASCT2 function is required.

Effect of ammonia, glutamine, and serum on calcineurin, p38MAPK-diP, GADD153/CHOP10, and CNTF in primary rat astrocyte cultures.

Primary astrocyte cultures were subjected to different experimental schedules using several concentrations of ammonia (1, 3, and 5 mM ammonium chloride), serum (2.5%, 5%, and 12%), and glutamine (0.5, 1, and 3 mM) to analyze the involvement of calcineurin (CaN) in hyperammonemia and its relation with p38MAPK-diP and ciliary neurotrophic factor (CNTF). We demonstrated that exposure to ammonia affects CaN content, and confirmed the ammonia-induced reduction of CNTF expression; however, the involvement of CaN and p38MAPK-diP in CNTF reduction could not be confirmed. On the contrary, an inverse relationship between CaN and p38MAPK-diP contents was clearly demonstrated. GADD153/CHOP10 content was always higher under hyperammonemic conditions as well as under glutamine exposure, probably due to the osmotic stress provoked by glutamine accumulation, which was induced after exposure to ammonia. Statistical analysis demonstrated significant interactions of ammonia and serum for CaN, GADD153/CHOP10 and CNTF contents. The exposure to glutamine also induced changes in GADD153/CHOP10 and CaN; however, CNTF content was not affected. In conclusion, CaN content was affected by exposure to ammonia and glutamine; the serum content of the culture medium had a strong influence on the astroglial response to ammonium chloride, and glutamine exposure only reproduced some of the ammonia effects.

Glutamine: commercially essential or conditionally essential? A critical appraisal of the human data.

Glutamine is a nonessential amino acid that can be synthesized from glutamate and glutamic acid by glutamate-ammonia ligase. Glutamine is an important fuel source for the small intestine. It was proposed that glutamine is necessary for the maintenance of normal intestinal morphology and function in the absence of luminal nutrients. However, intestinal morphologic and functional changes related to enteral fasting and parenteral nutrition are less significant in humans than in animal models and may not be clinically significant. Therefore, it is unclear whether glutamine is necessary for the preservation of normal intestinal morphology and function in humans during parenteral nutrition. It was suggested that both glutamine-supplemented parenteral nutrition and enteral diets may pre-vent bacterial translocation via the preservation and augmentation of small bowel villus morphology, intestinal permeability, and intestinal immune function. However, it is unclear whether clinically relevant bacterial translocation even occurs in humans, much less whether there is any value in the prevention of such occurrences. Results of the therapeutic use of glutamine in humans at nonphysiologic doses indicate limited efficacy. Although glutamine is generally recognized to be safe on the basis of relatively small studies, side effects in patients receiving home parenteral nutrition and in those with liver-function abnormalities have been described. Therefore, on the basis of currently available clinical data, it is inappropriate to recommend glutamine for therapeutic use in any condition.

Gamma-glutamyl transpeptidase-dependent mutagenicity and cytotoxicity of gamma-glutamyl derivatives: a model for biochemical targeting of chemotherapeutic agents.

Many carcinomas in humans are rich in gamma-glutamyl transpeptidase (GGT), a plasma membrane enzyme that reacts with extracellular substrates. Thus, biochemical targeting of chemotherapeutic agents may be achieved by converting anticancer drugs into their gamma-glutamyl derivatives. Chemical conversion of phenylhydrazine (PH) and biochemical modification of daunomycin (DM) into their gamma-glutamyl derivatives gamma-glutamyl phenylhydrazine (GGPH) and gamma-glutamyl DM (GGDM) resulted in the abolishment of their mutagenicity and cytotoxicity, as judged by decreased viability and increased mutant yields in cultures of several Salmonella Ames strains. Commercial gamma-glutamyl-p-nitroanilide (GGPNA) was not toxic or mutagenic. Mutagenicity and/or cytotoxicity of these gamma-glutamyl derivatives were restored upon reaction with GGT, with concomitant release of PH, and p-nitroaniline (PNA). The GGT-dependent release of DM from GGDM was demonstrated by thin layer chromatography (TLC), spectral analysis, and specific mutagenicity. Mutagenicity and/or cytotoxicity of gamma-glutamyl derivatives increased in the presence of glycylglycine, a GGT activator, and decreased in the presence of serine-borate, a GGT inhibitor. GGDM retained considerable DNA binding capacity. Its inability to kill and mutagenize was due to altered transport properties. The results are compatible with the notion that gamma-glutamylation is a feasible method for biochemical targeting of drugs containing a primary amino group to GGT-rich tumors.

Effects of L-glutamate on viabilities of cultured diploid skin fibroblasts and lymphocytes. Increased toxicity not observed in Huntington's disease.

The toxicity of L-glutamic acid toward cultured fibroblasts and lymphocytes from age-matched, sex-matched controls, patients with Huntington's disease (HD) and patients with other diseases has been studied. In an initial non-blinded study, L-glutamic acid (15 mM) exerted a significant toxic effect on HD fibroblasts, decreasing viability by approximately 60% after 48 h exposure. The magnitude of the toxic effect on HD fibroblasts as a group was significantly different from the mean effect on the normal control group (P less than 0.003) and non-HD control group (P less than 0.004). However, there was variability in the sensitivity of a given fibroblast culture to L-glutamate. This toxic effect was also seen in several normal control and non-HD control fibroblasts. In a second blinded study using cultured fibroblasts from HD patients and age-matched, sex-matched controls, we were unable to distinguish between HD and control cultures. No difference in L-glutamate toxicity was observed between control and HD lymphocytes in short-term cultures. We conclude that the toxicity of L-glutamate is not specific for HD cells and that this experimental approach will be of little value in identifying cells from patients with HD.

Excessive glutamine sensitivity in Alzheimer's disease and Down syndrome lymphocytes.

In addition to clinical and neuropathological similarities between Alzheimer's disease and Down syndrome there are genetic and biochemical data which suggest common disease mechanism. Using an in vitro assay examining variations of the mitotic index in the presence or absence of various inhibitors or metabolites of purine and/or pyrimidine synthesis, we studied 19 Alzheimer disease patients and 16 patients with both Down syndrome and Alzheimer type dementia. A highly significant decrease in mitotic index in the presence of exogenous glutamine was noted in patients presenting an Alzheimer type dementia with or without associated Down syndrome. These findings suggest that glutamine sensitivity or some dysregulation of the glutamine/glutamate pathway may play a role in the pathogenesis of Alzheimer's disease. If these findings are confirmed, they would have important implications in the development of preventive strategies.

Amplification of glutamate-induced oxidative stress.

Glutamate is a ubiquitous neurotransmitter which causes excess neuronal excitotoxicity and neurodegenerative insults such as stroke, trauma and seizures. A salient feature of the activation of glutamate receptors is the induction of oxidative burst. Moreover, glutamate stimulates Ca2+ influx and translocates protein kinase C (PKC). PKC mediates cellular processes mediated via phosphorylations which may be essential for oxidative burst in many cells. Subsequent oxidative stress may be a causal factor of neurodegenerative diseases. Increased glutamate release and oxidative burst may thus both be essential in the cascade of events leading to neuronal damage. Glutamate may also mediate neurotoxic effects of environmental toxic agents such as lead which amplify glutamate excitotoxicity. In these interactions, excessive activation of glutamate receptors and oxidative burst may converge into a common pathway leading to cell death through a cascade involving PKC or other protein important in oxidative burst in neurons.

Glutamine infusion during ischemia is detrimental in a murine gut ischemia/reperfusion model.

BACKGROUND: Gut ischemia/reperfusion (I/R) frequently occurs in clinical settings as a result of disproportionate splanchnic hypoperfusion during shock. Glutamine (GLN) supplementation of total parenteral nutrition (TPN) before gut I/R improves survival after gut I/R compared with standard TPN. However, it is unknown whether GLN treatment after the occurrence of the insult is beneficial or not. The aims of this study were to examine effects of GLN infusion during gut ischemia on survival, myeloid cell (neutrophils + monocytes) activation, and vascular permeability in organs. METHODS: Male Institute of Cancer Research (ICR) mice were randomized to control and GLN groups. After IV cannulation, mice underwent 90 (experiments 1 and 2) or 60 (experiment 3) minutes of gut I/R. Control mice received normal saline infusion at 1 mL/h for 60 minutes during ischemia, whereas the GLN group was given 3% GLN solution. In experiment 1, survival rates were monitored for 72 hours (n = 25). In experiment 2, peripheral blood was obtained at 2 or 4 hours after reperfusion (n = 17). Reactive oxygen intermediate (ROI) production by myeloid cells was determined by flow cytometry using dihydrorhodamine 123 with or without phorbol myristate acetate stimulation. Expression of CD11a and CD11b on myeloid cells was also measured. Myeloperoxidase (MPO) activity in the lung was evaluated. In experiment 3, vascular permeability in organs was measured using Evans blue at 2 or 4 hours. RESULTS: In experiment 1, survival time in the GLN group was significantly reduced compared with the control group (p = .02, log-rank test). The survival rates were 92% (12/13) and 42% (5/12) for the control and GLN groups at 12 hours (p = .01) and 38% (5/13) and 0% (0/12) at 48 hours (p = .02), respectively. In experiment 2, ROI production was significantly higher in the GLN group than in the control group after PMA stimulation both at 2 and 4 hours. CD11b expression was significantly higher in the GLN group than in the control group at 4 hours. There was no difference in pulmonary MPO activity at either time point. In experiment 3, GLN infusion significantly increased hepatic vascular permeability compared with saline infusion at 4 hours. CONCLUSIONS: GLN infusion during ischemia is detrimental for survival after gut I/R. A possible mechanism is excessive priming of myeloid cells caused by GLN infusion. Timing of GLN administration is critical for outcome after gut ischemic insult.

Oral subchronic and genotoxicity studies conducted with the amino acid, L-glutamine.

L-Glutamine is an abundantly occurring amino acid that serves numerous nutritional and physiological functions. It has current and potential applications as a therapeutic agent, dietary supplement, food ingredient, and in animal nutrition. To assess the safety of supplemental L-glutamine, a bacterial reverse mutation assay, in vitro chromosomal aberration assay, and a 13-week toxicity study were conducted. L-Glutamine showed no mutagenic activity in the bacterial reverse mutation assay, and did not induce chromosomal aberrations in Chinese hamster lung fibroblast cells in the in vitro chromosomal aberration assay. In the 13-week toxicity study, Sprague-Dawley rats (10/sex/group) were fed diets containing 0, 0.5, 2.5, or 5.0% L-glutamine. No deaths occurred, and no significant differences in body weights, body weight gains, ophthalmological findings, urinalysis parameters, or organ weights were observed between L-glutamine-fed rats and their respective controls. No toxicologically relevant effects on hematological or blood biochemical parameters were observed. Macroscopic and microscopic effects occurred at low frequency but were not associated with a dose-response relationship. Based on the results of the study, the no-observed-adverse-effect-level was determined to be 5.0% L-glutamine in the diet, the highest concentration tested (equivalent to 3832 and 4515 mg/kg body weight/day in male and female rats, respectively).

Glutamine as a mediator of ammonia neurotoxicity: A critical appraisal.

Ammonia is a major neurotoxin implicated in hepatic encephalopathy (HE). Here we discuss evidence that many aspects of ammonia toxicity in HE-affected brain are mediated by glutamine (Gln), synthesized in excess from ammonia and glutamate by glutamine synthetase (GS), an astrocytic enzyme. The degree to which Gln is increased in brains of patients with HE was found to positively correlate with the grade of HE. In animals with HE, a GS inhibitor, methionine sulfoximine (MSO), reversed a spectrum of manifestations of ammonia toxicity, including brain edema and increased intracranial pressure, even though MSO itself increased brain ammonia levels. MSO inhibited, while incubation with Gln reproduced the oxidative stress and cell swelling observed in ammonia-exposed cultured astrocytes. Recent studies have shown that astrocytes swell subsequent to Gln transport into mitochondria and its degradation back to ammonia, which then generates reactive oxygen species and the mitochondrial permeability transition. This sequence of events led to the formulation of the "Trojan Horse" hypothesis. Further verification of the role of Gln in the pathogenesis of HE will have to account for: (1) modification of the effects of Gln by interaction of astrocytes with other CNS cells; and (2) direct effects of Gln on these cells. Recent studies have demonstrated a "Trojan Horse"-like effect of Gln in microglia, as well as an interference by Gln with the activation of the NMDA/NO/cGMP pathway by ammonia as measured in whole brain, a process that likely also involves neurons.

Effects of dietary glutamine supplementation on lung injury induced by lipopolysaccharide administration.

Acute lung injury (ALI) is a critical syndrome associated with respiratory dysfunction, and neutrophils are considered to be central to the pathogenesis of ALI. This study investigated the effects of glutamine (Gln) on neutrophil recruitment in a model of lipopolysaccharide (LPS)-induced ALI. C57BL/6 mice were fed a standard diet either with casein as the nitrogen source or with 25% of total nitrogen replaced by Gln. After 10 days, intratracheal instillation of LPS was used to induce ALI. Mice were killed at 0, 6, 12, and 24 h after LPS administration (n = 10/group). Bronchoalveolar lavage fluid and lung tissues were collected for further analysis. The results showed that, compared with the control group, lipid peroxide levels in the lungs were higher at 12 and 24 h after LPS administration in the Gln group. CXC chemokines as well as tumor necrosis factor-alpha were significantly elevated and reached peaks at 6 h in the Gln group, which was earlier than in the control group. Histopathological findings showed that the thickening of alveolar septal space was extensive in the Gln group 24 h and 2 wk after LPS. Also, greater amounts of collagen had accumulated in lung tissue in the Gln group. This study indicates that dietary Gln administration resulted in higher inflammatory cytokine production, with more neutrophils recruited at the early stage of ALI. These results were consistent with the histopathological findings that Gln supplementation causes more severe interstitial inflammation and fibrosis in a model of ALI induced by LPS.

Risk assessment for the amino acids taurine, L-glutamine and L-arginine.

Taurine, glutamine and arginine are examples of amino acids which have become increasingly popular as ingredients in dietary supplements and functional foods and beverages. Animal and human clinical research suggests that oral supplementation of these amino acids provides additional health and/or performance benefits beyond those observed from normal intake of dietary protein. The increased consumer awareness and use of these amino acids as ingredients in dietary supplements and functional foods warrant a comprehensive review of their safety through quantitative risk assessment, and identification of a potential safe upper level of intake. The absence of a systematic pattern of adverse effects in humans in response to orally administered taurine (Tau), l-glutamine (Gln) and l-arginine (Arg) precluded the selection of a no observed adverse effect level (NOAEL) or lowest observed adverse effect level (LOAEL). Therefore, by definition, the usual approach to risk assessment for identification of a tolerable upper level of intake (UL) could not be used. Instead, the newer method described as the Observed Safe Level (OSL) or Highest Observed Intake (HOI) was utilized. The OSL risk assessments indicate that based on the available published human clinical trial data, the evidence for the absence of adverse effects is strong for Tau at supplemental intakes up to 3 g/d, Gln at intakes up to 14 g/d and Arg at intakes up to 20 g/d, and these levels are identified as the respective OSLs for normal healthy adults. Although much higher levels of each of these amino acids have been tested without adverse effects and may be safe, the data for intakes above these levels are not sufficient for a confident conclusion of long-term safety, and therefore these values are not selected as the OSLs.